Abstract

In an in vitro incubation the treatments in a 2*2 factorial arrangement
were: rumen fluid from cattle previously fed biochar
(BA), rumen fluid from cattle previously fed biochar + biochar added to
the substrate (BA+BC), rumen fluid from cattle not previously fed biochar (NBA)
and rumen fluid from cattle not previously fed biochar + biochar added to
the substrate (NBA+BC). There were 4 replications of each treatment. The
substrate contained (DM basis): 70% cassava root meal, 26.5-28% cassava
leaf meal and 2% urea. In treatments BA+BC and NBA+BC the biochar was added at
1.5% of the substrate DM. These ingredients were mixed together in the
incubation flask to which was added 480 ml of buffer solution and 120 ml of
strained rumen fluid taken by stomach tube from cattle fed cassava root, cassava
foliage and urea and either biochar (0.62% of diet DM) (for BA treatments) or no
biochar (treatments NBA).

Gas production and percentage substrate DM solubilized were increased, and
percent methane in the gas was reduced, when: (i) the rumen fluid in the
incubation flask was taken from cattle adapted to 0.62% biochar in their diet
(DM basis) over a 4 month period; and (ii) when biochar was added to the
incubation medium at 1.5% of DM. There were additive effects on methane
reduction when rumen fluid from adapted cattle was combined with biochar added
to the incubation medium.

Introduction

In a previous report from our laboratory (Leng et al
2012), we showed that addition of 0.62% biochar to the cassava root and cassava
leaf meal diet, fed to local “Yellow” cattle, resulted in decreased production
of methane in eructated gases. In the present experiment we tested the
hypothesis that the rumen fluid from the cattle that had been fed biochar would
retain properties conducive to decreasing methane production when added as
inoculum to an in vitro
incubation with cassava root and cassava leaf meal supplemented with urea as the
substrate.

Data collection and measurements

The gas volume was read from the collection bottles
directly after 24 hours and the percentage of methane in the gas measured using
a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK). Three
samples were measured from each collection bottle.At the end of the incubation the residual
insoluble substrate in the incubation bottle was determined by filtering the
contents through several layers of cloth that
retained particle sizes to at least 0.1mm and then this was dried (100 °C for 24
hours) and weighed.

The data were analyzed by the General Linear Model (GLM)
option in the ANOVA program of the Minitab (2000) Software. Sources of variation
in the model were: Replictates, Biochar, Rumen fluid source, Interaction Biochar*Rumen
fluid source and error.

Results and discussion

Gas production and percentage DM solubilized were increased, and percent methane
in the gas was reduced, when: (i) the rumen fluid in the incubation was taken
from cattle adapted to 0.62% biochar in their diet (DM basis) over a 4 month
period; and (ii) when biochar was added to the incubation medium at 1.5% of DM
(Table 2 and Figures 1 and 2). There were additive effects on methane reduction
when rumen fluid from adapted cattle was combined with biochar added to the
incubation medium (Figure 3).

Table 2. Mean
values for gas volume, methane percentage in the gas, DM solubilized and
methane per unit DM solubilized in an in vitro incubation with
cassava root and leaf meal using rumen fluid from cattle adapted to
biochar in the diet or not adapted, and with addition or not of
biochar to the incubation medium

Figure 2.
Effect of biochar (with or without) in the substrate in an in
vitro incubation with cassava root meal, cassava leaf meal and
urea and with rumen fluid from cattle adapted or not to biochar in
the diet

Figure 3.
Effect of adaptation to biochar in the diet, and of addition of biochar to the substrate, on percent reduction in methane production
(ml methane/g DM solubilized) in an in vitro incubation of
cassava root meal with cassava leaf meal and urea

Discussion

The studies now presented confirm that
biochar produced from rice husk at high temperature when added to an anaerobic
fermentative system using rumen fluid lowers the net production of methane. The
mechanisms for this net reduction are not explained by any of the studies so far
undertaken
and we provide below some speculative suggestions of how this might be
accomplished in order to stimulate discussions in this area, particularly by
young scientists.

Biochar provides a large
surface area for mixed microbial communities and undoubtedly, in the presence
of organic matter, biochars become impregnated with microorganisms (Anonymous
2012).

In these studies a mixture of high
starch and high fibre feed has been used as substrate for microbes in or from
the rumen. Digestion of complex substrates in the rumen requires the coordinated
activities of a number of different microbial species and is most efficient
when these microbes are components of communities, self-organised within a
structured biofilm (Wang and Chen 2009). Association and attachment to feed
particles by ruminal organisms is rapid (Cheng et al 1980) occurring within
five minutes of feed entering into rumen fluid (Bonhomme 1990). Ruminal
organisms attach to plant derived feed particles containing mostly structural
carbohydrates, and usually adhere through an extensive glycocalyx which encloses
the bacterial colonies. These commence structural plant degradation by secreting
enzymes, which then hydrolyse the exposed structural components of fibrous plant
parts (see Cheng et al 1980). The hydrolytic products of these initiating bacteria attract
other species with particular substrate requirements and in turn produce an
endogenous extra-cellular polymeric substance which forms the matrix for biofilm formation to
which microbial populations are attracted and grow in a structured or layered
biofilm (Costerton 2007). These multi species biofilms are either positioned on the external surface of
the plant tissue, or certainly with cereal grain particles, develop internally as substrate is solubilised and utilised (McAllister et al 1994) . In
these layered structures sequential degradation of both complex structural
carbohydrates and more readily fermentable starches/sugars occurs through a train of
microbes that complement one another in that the end products of the energy
metabolism of one group provide substrate for other closely closely associated
microbial colonies. As an example of this the cellulolytic organism
Fibrobacter succinogenese has the capacity to hydrolyse a number of complex
carbohydrates but only uses cellulose and its hydrolytic products in its energy
metabolism (Suen et al 2011). In doing so it strips other recalcitrant
structural carbohydrates to soluble components (mainly soluble sugars) providing
enzymic
access to the cellulose fibres they surround . The soluble mono and
disaccharides not used by F succinogneses then appear to diffuse from
the surface of the plant materials and are likely to be degraded further by
other microbes in the outer components of the biofilm matrix with the production
of VFA and ATP
and reduction of cofactors . Overall the rate of the fermentativeprocess depends on the rate of regeneration of reduced
cofactors and the release of hydrogen.
However, a requisite for continuing fermentation is a low partial pressure of
hydrogen since hydrogen build up would inhibit hydrogenase activity resulting
in lowered levels of NAD, NADP and FAD which slows or inhibits fermentation (see Wolin 1979; McAllister and Newbold 2008)
and this is normally controlled by the rate of methanogenesis.

Ruminal microorganisms can be
functionally described as four sub populations: (i) those associated with
ruminal fluid; (ii) those loosely attached to the feed particles
probably in the outer layer of the biofilm; (iii) those
tightly bound to the particle surface: and (iv) those that remain in the
biofilm within feed particles (see Wang and McAllister 2002). The rumen fluid
used in the incubation procedures is likely to have a population of microbes
different to the mixed digesta but the rapidity of attachment and biofilm growth
(Cheng et al 1980) suggests that these will be a source of inoculating organisms and will reflect
therefore the population densities in the rumen. Thus it is useful to study the
actions of rumen fluid from animals adapted to biochar and unadapted as the
differences in rumen conditions should be reflected in the changes particularly
of net methane production.

In these studies we are interested in
seeing whether there is benefit in mitigating enteric methane production by
increasing the inert surface areas where microbes may come together for mutual
feeding benefits.

The hypothesis developed from the
companion paper (Leng et al 2012) is that solubilised feed materials and
hydrogen diffusing from the particles being digested are partially
or totally taken up as
they diffuse towards the bulk fluid in the rumen and that this will be aided by
inert materials that provide surfaces either closely associated with the feed
particle and biochar particles or by biochar suspended in the fluid.

Rumen fluid obtained in these studies is
not representative of rumen digesta being lower in particles where it could be expected
most of the effects of biochar will be exerted However, we also
anticipated that since the colonisation of feed is extremely rapid, that if biochar increased the
population density of microbes in the digesta, that the levels of these would
be amplified in the liquor/small
particle mix used in the incubation medium.

The results overall indicate that
adapted rumen fluid reduced net methane production and increased the rate of
substrate solubilisation perhaps consistent with a larger population in rumen
digesta of the consortia that oxidise methane. The additive effect of biochar in
vitro with rumen fluid from animals adapted to biochar can also be hypothesised
to be due to an initial higher population of these same organisms in the
rumen fluid from adapted
animals. The response to added biochar to rumen fluid from non adapted sources
which is higher than that from adapted rumen fluid without biochar suggests that
the population dynamics of these were lower and that the activity had been lower
in the absence of the biochar.

It is clear that
biochar in a diet/substrate alters the population mix of microbes in the
digesting medium. Its ability to be impregnated with microbes is probably
the major association here and it is tentatively suggested that the biochar
creates a house (habitat) for the association of microbes that either more
efficiently ferment feed materials or possibly where methane oxidation is facilitated by
bringing together methanogenic Archae and a methanotrophic consortia (Knittel
and Boetius 2009). Such an association could result in an increased
oxidation of methane by a bolstered population density of methanotrophs in biochar adapted
rumens,where
normally these populations are extremely small (Kajikawa and Newbold 2003).
It appears that the anaerobic methane oxidation is carried out by an as yet
unknown consortia of Archae /bacteria and
it is possible that the lowering of methane by biochar addition to the rumen is solely a result of the increase in potential
habitat for this consortium. However, in the biodigester with added biochar, the
biochar has been observed after 4 weeks to become impregnated with, in
particular, methanosarcina-like species
(Anonymous 2012) so also providing habitat for both
methanogenic and methanotrophic organisms in very close association. To complete
these speculations we put forward the concept
that reduced methane production could be a result of carbon and sulfur back flux during
anaerobic microbial oxidation of methane and coupled sulfate reduction that has
been recently described by Holler et al (2012) where the methanogenic Archae
were
responsible for both production of methane and some oxidation of methane by
reversal of methanogenesis.

Conclusions

Gas production and percentage substrate DM solubilized were increased, and
percent methane in the gas was reduced, when: (i) the rumen fluid in the
incubation flask was taken from cattle adapted to 0.62% biochar in their diet
(DM basis) over a 4 month period; and (ii) when biochar was added to the
incubation medium at 1.5% of DM.

There were additive effects on methane
reduction when rumen fluid from adapted cattle was combined with biochar added
to the incubation medium.

Acknowledgements

The authors acknowledge support for this research from the MEKARN project financed by Sida. Special thanks to Mr Sengsouly Phongphanith, Mr Khamphout Thammavong and Mr Touvieu Xaiker, who provided valuable help in the farm. We also thank the Department of Animal Science, Faculty of Agriculture and Forest Resources, Souphanouvong University for providing infrastructure support to carry out this research.

Inthapanya S, Preston T R and Leng R A 2011
Mitigating methane production from ruminants; effect of calcium nitrate as
modifier of the fermentation in an in vitro incubation using cassava root as the
energy source and leaves of cassava or Mimosa pigra as source of protein.
Livestock Research for Rural Development. Volume 23, Article #21.
http://www.lrrd.org/lrrd23/2/sang23021.htm